The present invention relates to aminopeptidases, their production and their industrial use.
Hydrolysis of proteins is of great importance, particularly in the food industry. It generally consists in transforming high molecular weight proteins (e.g. casein, gluten, gelatin, soy protein) into small fragments (oligopeptides or amino acids). These transformations may be carried out under highly acidic or highly alkaline conditions, which can often be energy consuming and requires the use of aggressive chemicals.
Protein degrading enzymes are preferred because they are less polluting and capable of working under mild conditions which prevents racemization of amino acids. These enzymes are classified into endoproteases and exopeptidases. Endoproteases cleave high molecular weight proteins into oligopeptides, whereas exopeptidases release amino acids from high molecular weight proteins or protein fragments.
Both types of enzyme are generally necessary to produce protein hydrolysates. The endo/exo ratio has to be varied according to the application. To obtain a strong liquefaction of a complex protein, endo enzymes are required to provide the major hydrolysis contribution. Conversely, to produce specific amino acids or peptides from a complex protein without destroying its physical properties (elasticity, foaming property, texture property) exo enzymes with low endo activity will be preferred.
Many microorganisms are able to produce endoprotease and exopeptidases. The endo/exo ratio is mainly dependent on culture conditions (1-3) and on downstream processing (4).
In the food industry, Aspergilli have been widely used for a long time and are therefore easily in conformity with regulations in many countries all over the world. Among Aspergilli, Aspergillus niger is the most widely used species in the food industry. Whereas endoprotease (5-7) and carboxypeptidase (8-10) from Aspergillus niger have been described, the production of aminopeptidase from Aspergillus niger has up to now not been known.
The present invention discloses aminopeptidases from selected Aspergillus niger strains and a method for producing them from appropriate cultures of Aspergillus niger. These aminopeptidases have an optimal activity at a pH in the range 6-8 and at a temperature in the range 50-60.degree. C. Moreover, under appropriate culture conditions, aminopeptidases can be produced by selected Aspergillus niger strains substantially free of endoprotease. By "substantially free of endoprotease" is meant without detectable, or at least without a substantial amount of, endoprotease. Thus, surprisingly, it has been found that by culturing selected Aspergillus niger strains, a fermentation broth filtrate or liquid concentrate thereof can be obtained containing small amounts of endoprotease, but containing a high amount of aminopeptidase activity. Thus, a cell-free preparation of Aspergillus niger aminopeptidase may, for example, advantageously have at least 10 times more aminopeptidase activity, preferably at least 30 times more aminopeptidase activity, than endoprotease activity.
In view of recent changes in the nomenclature of black Aspergilli, the term Aspergillus niger is herein defined as including all (black) Aspergilli that can be found in the Aspergillus niger Group as defined by Raper and Fennell (1965, In: The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp 293-344). Similarly, also for the other Aspergillus species we will refer to the Aspergillus groups as defined by Raper and Fennell supra, thereby including all species and variants included in a particular group by these authors.
A phenylalanine-aminopeptidase (Phe-AP) and a leucine-aminopeptidase (Leu-AP) have been identified and characterised in such preparations, the phenylalanine-aminopeptidase making the major contribution to the total aminopeptidase activity. It will be appreciated, however, that the invention also extends to preparations of functional derivatives of Aspergillus niger aminopeptidases substantially free of endoprotease.
Thus, in one aspect, the present invention provides a cell-free preparation of Aspergillus niger aminopeptidase or a functional derivative thereof which is substantilly free of endoprotease.
In a further aspect, the present invention provides a process for preparing such an enzyme preparation which comprises fermenting an Aspergillus niger strain capable of producing aminopeptidase, e.g. Aspergillus niger NRRL 3112 or Aspergillus niger CBS 115.39, under conditions whereby aminopeptidase is produced in the fermentation broth, filtering the fermentation broth and optionally concentrating the filtrate thus obtained, e.g. by ultra-filtration (UF concentration). Preferably, concentration of the fermentation broth filtrate will be followed by addition of a stabilizing agent, preferably, for example, glycerol, e.g. at 50% (v/v). If desired, one or more aminopeptidases may be separated from the fermentation broth filtrate. The aminopeptidase is preferably substantially cell-free.
An aminopeptidase preparation according to the invention has several commercial applications. For example, such a preparation can be used advantageously for the preparation of baked products such as bread. 1 to 100 units Phe-AP, preferably 5 to 50 units Phe-AP per kg of dough results in an improved flavour and aroma for the baked product. Other uses can be found in food and feed applications such as cheese ripening, protein hydrolysates, debittering and yeast extract production.
Thus, in a still further aspect, the present invention provides a food product or food preparation intermediate containing a preparation of Aspergillus niger aminopeptidase or a functional derivative thereof as hereinbefore described.
5 to 500 Phe-AP, preferably 15 to 250 Phe-AP per 1000 liters of milk results in an improved taste, flavour aroma, consistency and texture of the cheese at an earlier stage during ripening of the cheese.
We found that aminopeptidase is capable of producing free amino acids in semi hard cheese. An enzyme composition containing the aminopeptidase in conjunction with acid protease e.g. from Mucor miehei was found to be capable of producing bitterness reduced cheese.
In the cheesemaking process aminopeptidase is initially liberating rapidly high quantities of cheese flavour generating free amino acids. Surprisingly we found that the initial liberation of the free amino acids (see Example 5) stopped and did not result in overripening of cheese. This is in contrast with a lot of prior art processes wherein by the action of proteolytic enzymes this liberation of amino acids continues and which might result in overripening. A possible explanation of this continued liberation might be the combined action of endo and exoproteases.
The coagulants used in cheese making generally perform a vital role in proteolysis during the ripening of the cheese, apart from the clotting activity they perform.
Traditionally all coagulants used in cheese making are enzyme preparations with one main enzymatic activity (protease) accompanied by some secondary enzymatic activities (in general also proteases), like for instance animal rennet in which chymosin and pepsin are predominantly responsible for both the coagulating action as well as for part of the proteolysis in cheese.
The same is true for microbial rennet from Mucor miehei and/or Endothia parasitica; in fact it is expected that the association of bitterness in cheeses made with this type of coagulants could depend on its secondary enzyme activities.
When making cheese, scientific experiments do often not take into account variations in quality of incoming cheese milk at commercial cheese production plants. The incoming milk can easily be contaminated with exogenous enzyme systems deriving from the cow or from contaminating microorganisms. These contaminants are frequently insufficiently inactivated by the pasteurisation treatments used for cheese milk.
Attempts to accelerate cheese ripening at commercial scale, for instance by simply raising the ripening temperature, often encounter the drawback that good quality flavour is enhanced for cheeses derived from impeccable lots of milk but that defective milk lots just as often result in enhancing of off-flavours yielding a negative net gain.
The impact of above mentioned contaminants on cheese making parameters is thought to be one for this assessment.
The secondary activities of coagulant enzymes are also to be held partially responsible for the amplification of the off-flavours produced in those occasions.
The use of conventional and existing blends of accelerated ripening enzymes, invariably mixtures of crude endo- and exopeptidases, provide a similar and additional risk; probably even the greatest of the three.
Therefore the aminopeptidase with standardised activity and free of endopeptidase activity is preferably used in combination with a standardised preparation of fermentation derived chymosin, with standardised and known endopeptidase activity and free from pepsin and/or other contaminating proteases.
Preferably also neutral protease is added more preferably neutral protease from Bacillus, more preferably from Bacillus subtilis, with standardised endopeptidase activity free from secundary and serine protease activities. Possible routes to obtain purified neutral protease are a concentration and purification process starting with a fermentation broth containing the neutral protease. Another possibility is the production in a transformed host, which system is designed for its selective production of the desired enzyme.
These enzyme compositions allow for the development of a cheese making process in which the acceleration of cheese maturation takes place under reduced risk of producing off flavours as associated with occasional fluctuations in milk quality and incidental variations in the cheese making process.